I am performing a sandwich competitive ELISA for PGF2alpha that was developed in our lab. I can not get the sensitivity below 25 pg/ml.

Average (B/Bo)50 pg/ml 70% 25 80%12.5 90%6.25 93%3.125 94%

We have tried increasing incubation times of the primary, standard/samples and secondary but that decreases sensitivity. Does anyone have any ideas that I can try? Would an HRP amplification kit help? I talked to the person who originallly developed the assay and she does not remember how she got her 3.125 standard value to be 80%, but she remembers it being hard. Thanks for any advice.

-tlp-

QUOTE (tlp @ Mar 12 2008, 07:41 AM)

I am performing a sandwich competitive ELISA for PGF2alpha that was developed in our lab. I can not get the sensitivity below 25 pg/ml.

Average (B/Bo)50 pg/ml 70% 25 80%12.5 90%6.25 93%3.125 94%

We have tried increasing incubation times of the primary, standard/samples and secondary but that decreases sensitivity. Does anyone have any ideas that I can try? Would an HRP amplification kit help? I talked to the person who originallly developed the assay and she does not remember how she got her 3.125 standard value to be 80%, but she remembers it being hard. Thanks for any advice.

I am new at this and for this reason I have been reading very carefully (am not a biologist...). I am trying to quantify for a Nerve Growth Factor and in the protocol I found that "acid treatment" can enhance the detection of my protein. I do not know if this applies to your sample. It is a simple procedure where you bring down the pH of what you want to analyze and then bring it back close to neutral. I read that it is to make the protein available for recognition by the antibodies by dissociating a noncovalent bond of a peptide from the molecule of interest. If you think this can be helpful I can give you more details.